The present invention relates to a thermostable enzyme with a three-dimensional crystal structure, and more particularly to a thermostable phytase with a 2.1 xc3x85 crystal structure comprising six external blades and six internal calcium binding sites, wherein each blade consists of 4 or 5 anti-parallel xcex2-strands.
Phytase is an enzyme that degrades phytic acid into phosphate, phosphate inositol and inositol. The phytase, a thermostable enzyme, is known to be effective in digestion and its long-lasting catalytic activity makes the enzyme very useful as an additive for a livestock feed.
Phytic acid takes approximately 50-70 weight % of the total phosphorous stored in grain used as livestock feeds; however, monogastric animals such as hens and pigs are hardly able to utilize plant phosphate due to the deficiency in phytase in their bodies and thus most of the phytic acid they intake through the feed becomes excreted without being digested and eventually flowed into a drinking water source thus resulting in serious environmental contamination. In addition, phytic acid can bind to other essential elements of animal body such as trace minerals (e.g., Ca, Mg, Mn, Zn and Fe), amino acids and vitamins, and those nutrients bound to the phytic acid are in turn excreted in the form of an insoluble phytate without being absorbed into the body; this would not only cause the loss of nutrients required by the animal body but also bring about the devastation of natural environment. Therefore, by providing livestock with a phytase-added feed we can expect the economic benefits by reducing the amount of inorganic phosphate in feeds, the improvement of the bioavailability of the trace amount of essential bioactive materials present in livestock, and the environmental protection from contamination due to the reduction of phosphate excreted by livestock.
Despite the importance of utilization of phytase in livestock, the production of an idealistic phytase has not been very successful. First, the phytases developed so far can degrade only a few of the six phosphates in a phytic acid into phosphorous and also the production of a phytase is not economical because the enzyme requires a long period of culturing time. Moreover, since the enzyme is not very compatible with the digestive physiology of livestock, the enzyme often loses its activity in the digestive tract when fed to livestock and thus its initial purpose serving as a phosphate degrader has been much limited.
In general, a phytase is known digested in gastric tracts and then the digested phosphate is subsequently absorbed in small intestine. Therefore, it has been strongly required for the phytase to have an anti-acid property to maintain its vital enzyme activity in the presence of a strong digestive enzyme secreted in the small intestine of livestock and also a neutral type phytase, which is known to be catalytically active in small intestine, is preferred in this respect. Moreover, considering that the pelleting is performed at high temperature when feeding livestock, a thermostable phytase is a prerequisite.
A various lines of intensive global studies about a phytase have been conducted guided mostly by the studies in Europe (A. H. J. Ullah et al., J. Agric. Food Chem. 42, 423-425 (1994); K. C. Ehrlich et al., Biochem. Biophys. Res. Commun. 195, 53-57 (1993); C. S. Piddington et al, Gene, 133, 55-62 (1993)). For example, there have been studies on the effects of a phytase on animals (L. G. Young et al., J. Anim. Sci., 71, 2147-250. (1993); X. G. Lei et al., J. Anim. Sci., 72, 139-143(1994); Z. Mroz et al., J. Anim. Sci., 72, 139-143 (1994)), the structure of the phytase (D. Kostrewa et al., Nat. Struct. Biol. 4, 185-90 (1997), and a different line of study (L. F. Johnson et al., Ann. NY Acad. Sci. 165, 526-532 (1969) has recently shown that a phytase derived from Aspergillus niger degrades a 3xe2x80x2 phytate prior to degrading any phosphate located in other positions.
The inventors of the present invention developed a novel phytase derived from B. amyloliquefaciens DS-11 that enables to degrade phytate regardless of the positions of phosphate groups in a phytate, which was found to be more suitable to the digestive physiology of livestock and also capable of maintaining its enzyme activity during the feed production when a high temperature is applied.
The novel phytase was deposited to the Genetic Engineering Center attached to Korea Research Institute of Bioscience and Biotechnology (KRIBB) and was assigned a depository number xe2x80x98KCTC 0231BPxe2x80x99. The inventors also filed applications with the same enzyme in PCT (PCT/KR 98/00056), U.S. Pat. No. (09/142,621), Canada (2,249,014), Japan (JP9-532479) and in Korea (97-10948).
Nevertheless, the recent identification of the three-dimensional crystal structure of a phytase can help us to initiate further researches on developing new version of a phytase with an improved thermostability and an increased enzyme activity.
The present invention relates to a thermostable phytase with a 2.1 xc3x85 crystal structure of a propeller shape comprising six blades and six internal Ca2+ binding sites. The six blades, each of which comprises 4 or 5 anti-parallel xcex2-strands, encompass the external structure of the above crystal while the six Ca2+ binding sites, 3 with high-affinity and the other 3 with low-affinity, are embedded internally. In addition, the above-mentioned Ca2+ binding motifs can be applied in synthesizing highly thermostable proteins and active sites identified by the elucidation of an enzyme""s three-dimensional crystal structure can help to design new enzymes having those active sites by employing the recent advanced technology of protein engineering.